Edible vaccines expressed in yeast for preventing and treating infectious diseases, including hepatitis B, in humans

ABSTRACT

In the invention described here, the approach is to formulate an edible vaccine based on N-terminal yeast surface display expression platform that producing  S.cerevisiae  EBY100/pYD5-preS2/S(adw) and  S.cerevisiae  EBY100/pYD5-preS2/S(adr) for protecting and treating human against hepatitis B virus (HBV) infection, suggesting that yeast surface display expression system expressing HBsAg antigen has potential as a prophylactic treatment for HBV in human via oral vaccination. The technology developed in this patent application can also be used to produce edible (oral) vaccines for preventing and treating other infectious diseases in human.

FIELD OF THE INVENTION

The present invention is for the composition of an edible vaccine basedon yeast surface display expressions for creating an edible vaccine thatprevents and treats infections in humans, protecting humans from beinginfected with hepatitis B virus (HBV). The present invention comprisesmainly a N-terminal yeast surface expression system and oral vaccinationin humans.

BACKGROUND OF THE INVENTION

Hepatitis B virus (HBV) causes most cases of hepatitis in China and theworld. Although the world now has the tools to prevent hepatitis B,there is no cure for the millions of people already infected. In otherword, currently available HBV vaccine has a role in preventing healthhuman from HBV infection, but there is no cure role. Further,conventional immune route such as injection is not suitable for thelow-income people in the third world. Unfortunately, there are nocommercial HBV vaccine with oral administration. The purpose of thisinvention is to establish yeast surface display platform to deliverpreventive and therapeutic HBV vaccines by oral administration.

The concept of edible vaccine was proposed by Prof. Dominic Lam andexecuted by him and his colleagues in early 1990s who first reported theexpression of hepatitis B virus surface antigen (HBsAg) in tomato.Edible vaccines will be more acceptable because of its oral rather thaninjectable route of application. In contrast, producing the vaccines inplants could reduce the cost to less than a penny per dose, and simplefast food processing like drying and grinding could createnon-perishable preparations without refrigeration. Further, Prof.Dominic Lam and his research team also focus on Lactococcus and yeastbased vaccines which are used to prevent avian influenza infection.

Yeast surface display technology has been extensively developed forapplication in preventing virus affection. Recently, Saccharomycescerevisiae (S.cerevisiae) surface display was used to develop H5N1 andZika virus vaccines. Currently, P.pastoris secretion system has beendesigned to express HBsAg of HBV, these commercial HBV vaccines havebeen used for preventing HBV infection. Unfortunately P.pastorissecretion system would not be used to produce edible HBV vaccines.Importantly, there are no attempts to develop preventive and therapeuticHBV vaccines with oral administration using S.cerevisiae display systemwhich is more efficient than P.pastoris for producing HBV vaccines.

Collectively, we propose this invention that S.cerevisiae surfacedisplay system can be used to develop preventive and therapeutic HBVvaccines. To address this invention, HBsAg of HBV adw and adr genotypesare investigated by S.cerevisiae N-terminal surface display platforms.

Although the mechanism underlying the interaction between HBV and hostcells remain unknown, adw preS2/S (309 kDa) and adr preS2/S (30.8 kDa)are generally considered major surface antigen proteins of HBV, whichare involved in the infection process as an attachment protein. This isthe primary reason why preS2/S protein has been used as an effectivecandidate target for potential HBV vaccines development.

Vaccination is currently the only method that can effectively stop thespread of HBV in humans. Conventional platform based on P.pastorissecretion system for HBV vaccine shows limits for producing ediblevaccines. In the present invention, we describe a new type of potentedible HBV vaccine based on yeast surface display system.

The present invention can provide an effective way to protect human fromHBV infection and cure already affected. It may also be used to produceedible vaccines for preventing and treating other infectious diseases inhumans.

SUMMARY OF THE INVENTION

The present invention is about an edible vaccine for preventing andtreating HBV infection in humans. The present invention describes that aN-terminal display plasmid, pYD5, to display preS2/S (adw) or preS2/S(adr) protein on the surface of S.cerevisiae EBY100 and detected byWestern blotting, immunofluorescence and flow cytometric assay. Therecombinant yeast after lyophilization is encapsulated by entericcapsule, followed by ELISA detecting antibody responses. The presentinvention suggests that yeast display expression system can be developedfor edible HBV vaccines for preventing and treating HBV infection.

The present invention contains 3 major parts: (i) the construction ofrecombinant yeast, (ii) the recombinant yeast is lyophilized and thenencapsulated by enteric capsule, (iii) antibody responses from thevaccinated humans is detected by ELISA.

DETAILED DESCRIPTION OF INVENTION

Construction of HBsAg Antigen Surface-Displayed Yeast Vaccines

The preS2/S gene (Gene accession No. U87732.1, 846 bp) from HBV adwsubtype will be PCR-amplified using specific primers and subcloned intopYD5 in-frame with the endogenous Aga2p signal peptide sequence. Theresultant shuttle plasmid pYD5-preS2/S will be transformed into E. coliDH5α. The plasmid pYD5-preS2/S will then be extracted from E. coli,purified and electroporated into competent S.cerevisiae EBY100 afterbeing linearized. Recombinant yeast transformants will be plated onselective minimal dextrose plates containing amino acids (0.67% yeastnitrogen base without amino acids (YNB), 2% glucose, 0.01% leucine, 2%agar, and 1M sorbitol). Trp⁺transformants will be selected after 3 daysof growth on the selective minimal dextrose plates.

The positive colonies are confirmed by genomic PCR. Recombinant S.cerevisiae EBY100/pYD5-preS2/S(adw) is cultured in YNB-CAA-Glu (0.67%YNB, 0.5 casamino acids, 2% Glucose) and induced in YNB-CAA-Gal (0.67%YNB, 0.5 casamino acids, 2% Galactose, 13.61 g/L Na₂HPO₄, 7.48 g/LNaH₂PO₄ and 5 g/L casamino acids) at 20° C. with shaking (250 rpm) forinducing VP28 surface display. S.cerevisiae EBY 100 carrying pYD5plasmid (S.cerevisiae EBY 100/pYD5) is served as a negative control forall the subsequent tests.

One more type of HBV vaccine will be constructed in this section:

S.cerevisiae EBY100/pYD5-preS2/S(adr)—preS2/S surface displayed yeastvaccine.

Note: The preS2/S gene (Gene accession No. AF036239, 834 bp) from HBVadr subtype.

Determining the Functional Display of HBsAg Antigen on Yeast Surface

This experiment is designed to validate the functional display of thepreS2/S antigen on yeast surface.

Western Blotting

1 OD₆₀₀(1 OD₆₀₀≈10⁷ cells) equivalent recombinant yeast will becollected at different time point post inducement with 2% galactose. Thesamples are washed three times with 500 μl of PBS, re-suspended in 50 μlof 6×SDS loading buffer (Bio-Rad, Hercules, Calif.), and boiled for 10min. The surface presented preS2/S will be extracted by heating 1 OD₆₀₀of S.cerevisiae EBY100/pYD5-preS2/S(adw) pellets at 95° C. in aBromophenol blue sample buffer supplemented with 5%-ME for 5 min. Thesamples were then resolved on a 4-15% SDS-PAGE gel (Bio-Rad), andtransferred to 0.45 μm nitrocellulose membranes (Bio-Rad). Afterblocking with 5% non-fat milk at room temperature for 2 h, the membranewill be incubated with polyclonal horse anti-Hepatitis B Virus surfaceantigen (Abeam) as primary antibody (1:500 diluted). After incubatedovernight at 4° C. and washed three times using PBS buffer, themembranes will be reacted to the secondary antibodies, horseradishperoxidase (HRP)-conjugated goat anti-horse IgG (1:5,000 diluted)(Sigma-Aldrich Co., St. Louis, Mo.) for 1 hour at room temperature. Thesignal will be generated using West Pico chemiluminescent substrates(Thermo Fisher Scientific Inc., Rockford, Ill.) and detected using aChemiDoc XRS System (Bio-Rad).

Glycosylation Analysis of Yeast Surface Displayed PreS2/S

PNGase F is obtained from New England Labs (Beverly, Mass.). RecombinantS. cerevisiae EBY100/pYD5-preS2/S(adw) will be cultured at 30° C. inYNB-CAA-Glu overnight and then induced at 20° C. in YNB-CAA-Gal for 72hours. 1 OD₆₀₀ equivalent cells will be collected, centrifuged, andwashed once in a PBS buffer. Cell pellets were denatured at 100° C. for10 min in a denaturing buffer included in the PNGase F reagent. Aportion of 1 μL of PNGase F (5,000 U) will be added to the denaturedprotein solution, followed by incubation at 37° C. for 1 hour accordingto the manufacturer's instruction. The treated samples will then besubjected to Western blotting analysis.

Immunofluorescence Microscopy

To determine preS2/S display on yeast surface, recombinant S. cerevisiaeEBY100/pYD5-preS2/S(adw) will be collected in a 24-hour interval over a72-hour time period after induction with galactose (2%). 1OD₆₀₀equivalent recombinant yeast will be collected and blocked with 5%non-fat milk in PBS for 1 hour, and incubated with polyclonal horseanti-Hepatitis B Virus surface antigen (1:500 diluted) at 4° C. for 1hour. After washing with PBS, the samples will be incubated with goatanti-horse IgG FITC conjugates (Sigma) (1:5,000 diluted) at roomtemperature for 1 h. The samples will be kept in dark until use. TheFITC labeled yeast will be examined under an inverted phase contrastfluorescence microscope.

Flow Cytometric Assay

After inducement with galactose (2%), 1 OD₆₀₀ equivalent recombinantyeasts will be collected over a 72-hour time period, with a 24-hourinterval, as described above. The cell samples will be washed threetimes with sterile PBS containing 1% bovine serum albumin (BSA) andincubated with polyclonal horse anti-Hepatitis B Virus surface antigen(1:500 diluted) at 4° C. for 1 hour, followed by reacting withFITC-conjugated goat anti-horse IgG (1:5,000) at 4° C. for 30 min. Thecell samples will be re-suspended in 500 μL of sterile PBS and will besubject to flow cytometric analysis using a BD FACS Aira III (BDBioscience, San Jose, Calif.). S.cerevisiae EBY100/pYD5 served as anegative control for the assay. These data will be used to ascertainwhich time point will be the best for collecting yeast vaccines thatpresent the highest level of preS2/S on their surface.

Note: The similar methods are used to determine the functional displayfor the following S.cerevisiae EBY100/pYD5-preS2/S(adr).

Lyophilization and Encapsulation

Recombinant yeast is lyophilized and then encapsulated by entericcapsule.

Optimization of Oral Immunization

HBV transgentic mice is used in this invention. A batch of mice wasdivided into five groups and 10 mice per group were selected. Each mouseorally vaccinated with one capsule containing recombinant yeast feedcontinuously for 3 days, boosted at day 15-18. The oral vaccinationexperiments were repeated three times.

ELISA Assay

Tenth day after the final vaccination, blood sample was isolated fromthe vaccinated mice.

Antibody responses of serum IgG was determined by enzyme-linkedimmunosorbent assay (ELISA) using recombinant HBaAg protein (2 μg/ml) asa coating antigen as described previously. Optical density (OD) wasmeasured at 405 nm using ELI SA plate reader. The IgG titer wasdetermined to be the lowest dilution with an OD greater than the mean ODof naïve controls plus 2 standard deviations.

Based on these results, we can evaluate and determine the strength anddegree of immune protection that can be provided by the yeast vaccines.Further, we can determine which vaccine provides the complete immuneprotection of human from HBV infection.

What is claimed is:
 1. A composition for preventing and treatinghepatitis B virus (HBV) infection in humans comprising a yeastexpression plasmid, pYD5, for N-terminal surface display, wherein therecombinant plasmid contains SEQ ID NO: 1 or SEQ ID NO:2.
 2. Thecomposition of claim 1, wherein yeast that includes recombinant plasmid.3. The composition of claim 2, wherein the said yeast is S.cerevisiaeEBY100.
 4. The composition according to claim 2 or 3, wherein the saidrecombinant yeast can be used for developing vaccine for preventing HBVinfection.
 5. The composition of claim 4, wherein the said vaccine isorally administrated.
 6. The composition according to claim 2 or 3,wherein the HBV vaccine formulation comprises recombinant yeast plusadditional component.
 7. The composition of claim 6, wherein the saidformulation is orally administrated.
 8. A composition as claimed inclaim 7, antibody responses from the vaccinated mice are detected byELISA.
 9. A method for constructing recombinant yeast comprising thestep of applying the composition in claim 1 or
 2. 10. A method fordelivering recombinant yeast comprising the step of applying thecomposition in claim 5, 6 or
 7. 11. A method for evaluating theprotective efficiency of recombinant yeast comprising the step ofapplying the composition in claim 8.